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Tracing the development of acute myeloid leukemia in CBL syndrome

Heiko Becker,1 Kenichi Yoshida,2,3 Nadja Blagitko-Dorfs,1 Rainer Claus,1

Milena Pantic,1 Mahmoud Abdelkarim1, Christoph Niemöller,1 Christine Greil,1

Björn Hackanson,1 Yuichi Shiraishi,4 Kenichi Chiba,4 Hiroko Tanaka,5 Satoru Miyano,4,5

Konstanze Döhner,6 Susanne Schnittger,7 Philipp Henneke,8,9 Charlotte M. Niemeyer,9

Christian Flotho,9 Dietmar Pfeifer,1 Seishi Ogawa,2,3 and Michael Lübbert1

1 Department of Medicine I, Medical Center - University of Freiburg, Freiburg, Germany;

2 Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto

University, Kyoto, Japan;

3 Cancer Genomics Project, Graduate School of Medicine, The University of Tokyo,

Tokyo, Japan;

4 Laboratory of DNA Information Analysis, Human Genome Center, Institute of Medical

Science, The University of Tokyo, Tokyo, Japan;

5 Laboratory of Sequence Analysis, Human Genome Center, Institute of Medical

Science, The University of Tokyo, Tokyo, Japan;

6 Department of Internal Medicine III, University of Ulm, Ulm, Germany;

7 MLL Munich Leukemia Laboratory, Munich, Germany;

8 Center of Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg,

Germany;

9 Center for Pediatrics and Adolescent Medicine, Medical Center - University of

Freiburg, Freiburg, Germany

Blood First Edition Paper, prepublished online February 3, 2014; DOI 10.1182/blood-2013-10-533844

Copyright © 2014 American Society of Hematology

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Corresponding author: Michael Lübbert, Department of Medicine I, Medical Center -

University of Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany, phone: +49 761

270 32790, fax: +49 761 270 36970, email: [email protected]

Running head: AML in CBL mutated hematopoiesis

Keywords: CBL syndrome, CBL mutation, acute myeloid leukemia, AML, juvenile

myelomonocytic leukemia

Supplemental data: The article is accompanied by supplemental data.

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Key Points

• The CBL syndrome may predispose to myeloid neoplasias other than JMML

• Whole-exome sequencing identifies mutations that possibly cooperate with mutant

CBL in AML development

Abstract

We describe the development of acute myeloid leukemia (AML) in an adult with CBL

syndrome due to a heterozygous de novo germline mutation in CBL codon D390. In the

AML bone marrow, the mutated CBL allele was homozygous after copy number-neutral

loss-of-heterozygosity and amplified through a chromosomal gain; moreover, an

inv(16)(p13q22) and, as assessed by whole-exome sequencing, 12 gene mutations

(e.g., in CAND1, NID2, PTPRT, DOCK6) were additionally acquired. During complete

remission of the AML, in the presence of normal blood counts, the hematopoiesis stably

maintained the homozygous CBL mutation, which is reminiscent of the situation in

children with CBL syndrome and transient juvenile myelomonocytic leukemia. No

additional mutations were identified by whole-exome sequencing in granulocytes during

complete remission. The study highlights the development of AML in an adult with CBL

syndrome and, more general, in genetically aberrant but clinically inconspicuous

hematopoiesis.

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Introduction

Preceding hematologic disorders are documented in a quarter of adults with acute

myeloid leukemia (AML).1 However, an unknown proportion of AMLs that apparently

arise de novo may have developed from undiscovered abnormal hematopoiesis.

Mutations in CBL, encoding an E3 ubiquitin ligase, are found in 10-20% of chronic

(CMML) or juvenile myelomonocytic leukemia (JMML) patients.2-7 Germline CBL

mutations cause the CBL syndrome that recapitulates features of other RAS-MAPK

pathway disorders and predisposes to JMML.8-10 In AML, CBL mutations are rare, but

associate with inv(16).11-14

Here, we describe the development of AML in an adult with CBL syndrome and JMML-

typical loss of wildtype CBL in bone marrow.

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Methods

Written informed consent of the patient included in the present study was obtained for

sample storage and analyses prior to sampling, as approved by the local ethics

committee. This study was conducted in accordance with the Declaration of Helsinki.

Karyotype, mutations in NPM1, FLT3 (tyrosine kinase domain, internal tandem

duplication), CEBPA and CBL, and CBFB-MYH11 expression relative to ABL1 were

assessed as described elsewhere.15-18 CBL mutated-to-wildtype allelic ratios were

determined using the PyroMark Q96MD (Qiagen), and chromosomal copy numbers

using CytoScanHD-arrays (Affymetrix). Data are deposited at

http://www.ebi.ac.uk/arrayexpress/ (E-MEXP-3997). Whole-exome sequencing was

performed as reported;19 variants were validated by Sanger sequencing. Methods are

detailed in the supplement.

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Results and discussion

Characteristics of the AML

A 40-year-old male was diagnosed with AML in June 2011. Pre-existing conditions were

hereditary spherocytosis (diagnosed in 1996), coagulopathy (low FVII, X, XII, XIII), atrial

fibrillation and hypocholesterolemia; a splenomegaly was considered consequence of

the spherocytosis. At AML diagnosis, the white blood cell count was 19,390/µl, with

approximately 30% blasts and 30% dysplastic monocytes (Supplemental Figure S1).

The marrow contained 50% CD117-positive blasts and 30% CD14-positive monocytes;

the karyotype was 46,XY,add(4)(q?31),inv(16)(p13q22)[21]/46,XY,inv(16)(p13q22)[1].

CBFB-MYH11 (type D) was detected with a ratio of 46.23 in blood. NPM1, CEBPA and

FLT3 mutations were absent.

The patient received “3+7” induction followed by dasatinib (ClinicalTrials.gov

NCT00850382). Six weeks after therapy start, complete remission (CR) was

documented. He received four consolidation courses with high-dose cytarabine. At last

follow-up (September 2013), he was in continuous CR, with no CBFB-MYH11

detectable.

Identification of a germline CBL mutation

Between the treatment courses, the patient’s monocyte counts rose to extraordinarily

high levels. Although monocytes were within normal limits after treatment and in blood

counts dating back to 1996 (Supplemental Table S1), this observation prompted the

question whether the patient had an underlying monocytic disorder.

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Since monocytosis is a hallmark of JMML and CMML, we examined the mutation status

of CBL exon 8 and 9 in blood collected at the AML diagnosis. We indeed found a

p.D390V-mutation, located in the frequently mutated RING finger domain. Assessing

the germline origin of the mutation, we also identified it in buccal mucosa and hair

follicles. We concluded that the patient had a previously undiagnosed CBL syndrome,

with the preexisting coagulopathy and atrial fibrillation being part of the phenotype

(Supplemental Table S2). 8-10

No CBL mutations were detected in blood of both parents, indicating de novo

occurrence in the patient’s germline. The patient has no siblings.

Zygosity of the CBL mutation

Copy number-neutral loss-of-heterozygosity (LOH) of the CBL-containing chromosomal

band 11q23.3 is common in children with CBL syndrome and JMML.8,10 11q-LOH was

also detectable in marrow mononuclear cells from our patient during AML. Moreover,

the 11q-LOH persisted in B-lymphocytes, granulocytes and monocytes collected later

during CR (Table 1, Supplemental Figure S2). This is reminiscent of the situation in

children with CBL syndrome and JMML whose myeloproliferation spontaneously

improves.8 Notably, the AML in our patient exhibited an additional gain of 11q-material,

indicating that the LOH had existed before the AML. In skin and T-lymphocytes, 11q

retained heterozygosity.

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To complement the LOH findings, we determined the allelic burden of the CBL mutation

by pyrosequencing (Table 1). In agreement with the LOH data, the mutation was

heterozygous in skin and T-lymphocytes but homozygous in AML cells and in

granulocytes, monocytes and B-lymphocytes collected during CR, where it remained

homozygous until last follow-up, underlining the stability of the genetically aberrant

hematopoiesis. Notably, LOH and pyrosequencing data suggested the presence of a

small fraction of T-lymphocytes also harboring the 11q-LOH (Table 1, Supplemental

Figure S2).

Identification of cooperating mutations by whole-exome sequencing

Similar to our patient, children with CBL syndrome and transient JMML feature normal

blood counts and persistent homozygous CBL mutation in their hematopoiesis.8 Little is

known about mechanisms that could be responsible for normal hematopoiesis despite

oncogenic features characteristic of JMML. We wondered whether this was associated

with the acquisition of mutations that overcome the myeloproliferative impact of the

homozygous CBL mutation. We therefore subjected granulocytes from CR and skin to

whole-exome sequencing, but identified no additional mutations.

We also performed whole-exome sequencing of AML cells to identify mutations that

were acquired during AML development, in addition to inv(16) and 11q-gain. We

detected somatic mutations in 12 genes (Table 2), three of which (i.e., CAND1, NID2,

PTPRT) were previously found mutated in AML.20-22 However, no gene has an

established role in leukemogenesis, e.g., as cooperating partner of mutant CBL or

CBFB-MYH11.

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Biologic impact of the CBL mutation

JMML features the formation of colonies at low concentrations of granulocyte-

macrophage colony-stimulating factor (GM-CSF).23 We observed no spontaneous

growth or hypersensitivity to GM-CSF of mononuclear cells collected from our patient

during CR (data not shown), which underlines the lacking or only subtle impact of the

homozygous CBL mutation on hematopoiesis. Moreover, granulocytes showed normal

production of reactive oxygen species and interleukin-8 to stimuli, and adhesion and

migration/chemotaxis were normal (data not shown).

In summary, we diagnosed a CBL syndrome in an adult, who, as observed in children

with CBL syndrome developing JMML,8,10 had lost the CBL wildtype allele in the bone

marrow. Whether this leads to overt JMML only under certain circumstances is not well

understood.24 Since the LOH persisted in the various hematopoietic cell lineages in our

patient, it likely conferred a clonal advantage at one point. Thus, the patient may have

indeed gone through a JMML or related hematologic disorder during infancy, which

spontaneously resolved and left behind normal blood counts. However, medical

information to support this assumption is unavailable. Following the hypothesis that

normal blood counts in our patient could be associated with the acquisition of mutations

counterbalancing the mutant CBL, we performed whole-exome sequencing but

identified no acquired mutations. On the background of the CBL mutation, the patient

developed AML through the acquisition of inv(16), gain of 11q-material and at least 12

gene mutations. The AML was erased by chemotherapy, again leaving behind a

hematopoiesis with homozygous CBL mutation.

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While the CBL syndrome is known to predispose to JMML, this is the first description of

of a different myeloid neoplasia occurring at adult age. It cannot be determined whether

the AML was mere coincidence, or due to a predisposition conferred by the CBL

mutation. However, the latter is supported by the specific gain of CBL-encoding 11q-

material and occurrence of inv(16), which associates with CBL mutations.11-14 If

substantiated by future studies, the association between CBL syndrome and AML

should be considered in clinical practice. CBL would then join other genes, e.g., RUNX1

or CEBPA, germline mutations in which were linked to a predisposition to AML.25

Overall, the case highlights the possibility of genetically aberrant hematopoiesis despite

normal blood counts and provides insight into myeloid neoplasias in the CBL syndrome.

Due to potential health problems associated with a CBL syndrome, germline analyses

may be generally warranted in younger adults with CBL-mutated neoplasias.

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Acknowledgements

We wish to express our thanks to the patient for continued and interactive participation

in the study. We also thank the German-Austrian AML Study Group (AMLSG), as the

patient was enrolled on an AMLSG clinical trial.

The research was supported in part by the Research Committee of the University

Freiburg, Germany [MER 785/10 (H.B.)], the EHA and ASH Translational Research

Training in Hematology (H.B.), the German Cancer Aid [DKH 110213 (B.H.)], the

Bundesministerium für Bildung und Forschung (P.H.) and the German Research

Foundation [DFG Lu 429/7-1, CRC 992-C04 (M.L.); DFG Fl 345/4-1, CRC 992-C05

(C.F.)].

Authorship

Contribution: H.B., K.Y., C.G., S.S., P.H., C.M.N, C.F., S.O., and M.L. contributed to the

design and interpretation of the study; H.B., N.B.-D., M.P., M.A., C.N., B.H., K.D., S.S.,

and C.F. carried out laboratory-based analyses; K.Y., R.C., Y.S., K.C., H.T., S.M., and

D.P. performed bioinformatic analyses; C.G., B.H., and M.L. were involved in patient

care; H.B., S.O., and M.L. wrote the manuscript, and all authors contributed to and

agreed on the final version.

Disclosure of conflicts of interest: S.S. declares part ownership of the MLL Munich

Leukemia Laboratory GmbH. The remaining authors declare no competing financial

interests.

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Correspondence: Michael Lübbert, Department of Medicine I, Medical Center -

University of Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany, email:

[email protected]

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Table 1. Chromosome 11q-aberrations assessed by single nucleotide polymorphism (SNP)-array and CBL D390V allele burden determined by sequencing in different cell populations.

Timepoint Cell type 11q-LOH* 11q-gain† CBL D390V allele burden

Diagnosis of AML BM MNCs Yes Yes 92.6%‡§

Complete remission of AML

PB Granulocytes Yes No 92.8% (90.1%-96.5%)‡|| PB Monocytes Yes No 92.2% (88.9%-95.5%)‡|| PB B-lymphocytes Yes No 83.3% (75.9%-94.1%)‡|| PB T-lymphocytes No¶ No 55.4% (52.4%-59.3%)‡||¶ Skin biopsy No No 48.6%‡§ Buccal mucosa ND ND Heterozygous# Hair follicle ND ND Heterozygous#

LOH indicates loss-of-heterozygosity; BM; bone marrow; MNCs, mononuclear cells; PB, peripheral blood; and ND, not determined. LOH data are also presented in Supplemental Figure S2. * LOH of chromosome 11 position 59764127-134942626; † Gain of chromosome 11 position 88486678-134938470; ‡ CBL D390V allele burden relative to combined D390V and wildtype alleles assessed by pyrosequencing; § average of measurements from one timepoint; || average and range of measurements at three time points during CR 5 to 19 months after AML diagnosis; ¶ data suggest small fraction of T-lymphocytes with 11q-LOH (purity of T-lymphocytes in pyrosequencing 97%-98%); # concluded from Sanger sequencing.

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Table 2. Gene mutations in AML acquired in addition to the germline CBL mutation and the chromosomal aberrations.

Gene Gene localization Mutation

ADAM12 10q26 NM_003474: c.665C>T, p.A222V* ARF3 12q13 NM_001659: c.302A>G, p.N101S CAND1 12q14 NM_018448: c.1750G>T, p.E584X CMIP 16q23 NM_030629: c.968C>T, p.T323M* DOCK6 19p13.2 NM_020812: c.5616_5617insCCG, p.R1872_K1873insP KIF14 1q32.1 NM_014875: c.1021G>A, p.V341I MIOX 22q13.3 NM_017584: c.673T>C, p.W225R* MYOCD 17p11.2 NM_153604: c.847G>A, p.D283N* NID2 14q22.1 NM_007361: c.955G>A, p.D319N PRSS16 6p21 NM_005865: c.1471C>T, p.R491C* PTPRT 20q12-q13 NM_007050: c.2531C>T, p.T844M* TMEM125 1p34.2 NM_144626: c.337G>A, p.D113N*

All mutations were identified by whole-exome sequencing of bone marrow MNCs from the AML. Their presence and somatic origin were validated by Sanger sequencing of AML and skin fibroblasts. The information on gene localization is based on Entrez Gene. * missense mutations that are “probably damaging” according to PolyPhen-2 (v2.2.2r398, HumDiv-trained model)

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